Bottom Line:
Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264.Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures.These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.

ABSTRACTX-ray crystallography provides excellent structural data on protein-DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein-DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein-DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.

gks291-F1: B-DNA docked to the DNA-bound structure of UNG (gray CÎ± backbone). (A) The 30 top-ranked B-DNA placements compared with bound DNA from 1SSP (blue phosphate backbone): 21 (yellow) at the active site; 6 (green) tightly clustered at a secondary site; 1 (magenta) between the active site and the secondary site; and 2 (orange) near the UNG N-terminus. (B) The larger active-site cluster (14 placements) replicates the UNGâ€“DNA active-site contacts found in the 1SSP complex, including insertion of Leu 272 (black) into the DNA minor groove. These dockings also show direct contact of the complementary strand with residues 210â€“220 (magenta). In all, the active-site strand has the same 5â€² to 3â€² direction as the crystallographic DNA, as indicated by red coloring of the 3â€²-ends. The UNG backbone is colored by the DXMS results (see Figure 3). (C) The 2000 top-ranked B-DNA placements, represented by their geometric centers (spheres), are concentrated over the active site (indicated by the crystallographic DNA, blue, right), at the secondary site (indicated by docked B-DNA, green, left), and between the two sites.

Mentions:
Docking calculations (Supplementary Methods) used a cubic grid 128 Ã… on a side with 1 Ã… grid spacing (about 2.1 million points). The DNA was centered at each grid point in 54â€‰000 distinct orientations, giving â‰ˆ108 billion placements of the DNA about UNG. The 2000 placements with the most favorable interaction energies, calculated as the sum of electrostatics and van der Waals intermolecular energy terms, were kept. These energies were mapped to the grid point at which the DNA was centered, allowing the distribution of the placements over the UNG to be visualized. The 30 top-ranked placements using coordinates from the UNGâ€“DNA crystallographic complex were analyzed by calculating the rmsd between the docked DNA and the crystallographic DNA. Calculation of rmsd is a poor method for clustering B-DNA placements (6,9), because lack of sequence recognition results in shifts along the DNA axis by one or more base pairs within the same cluster. Instead, the 30 top-ranked placements and the distribution of the 2000 top-ranked placements over the UNG surface were analyzed with computer graphics (Figure 1C and Supplementary Figure S2A).Figure 1.

gks291-F1: B-DNA docked to the DNA-bound structure of UNG (gray CÎ± backbone). (A) The 30 top-ranked B-DNA placements compared with bound DNA from 1SSP (blue phosphate backbone): 21 (yellow) at the active site; 6 (green) tightly clustered at a secondary site; 1 (magenta) between the active site and the secondary site; and 2 (orange) near the UNG N-terminus. (B) The larger active-site cluster (14 placements) replicates the UNGâ€“DNA active-site contacts found in the 1SSP complex, including insertion of Leu 272 (black) into the DNA minor groove. These dockings also show direct contact of the complementary strand with residues 210â€“220 (magenta). In all, the active-site strand has the same 5â€² to 3â€² direction as the crystallographic DNA, as indicated by red coloring of the 3â€²-ends. The UNG backbone is colored by the DXMS results (see Figure 3). (C) The 2000 top-ranked B-DNA placements, represented by their geometric centers (spheres), are concentrated over the active site (indicated by the crystallographic DNA, blue, right), at the secondary site (indicated by docked B-DNA, green, left), and between the two sites.

Mentions:
Docking calculations (Supplementary Methods) used a cubic grid 128 Ã… on a side with 1 Ã… grid spacing (about 2.1 million points). The DNA was centered at each grid point in 54â€‰000 distinct orientations, giving â‰ˆ108 billion placements of the DNA about UNG. The 2000 placements with the most favorable interaction energies, calculated as the sum of electrostatics and van der Waals intermolecular energy terms, were kept. These energies were mapped to the grid point at which the DNA was centered, allowing the distribution of the placements over the UNG to be visualized. The 30 top-ranked placements using coordinates from the UNGâ€“DNA crystallographic complex were analyzed by calculating the rmsd between the docked DNA and the crystallographic DNA. Calculation of rmsd is a poor method for clustering B-DNA placements (6,9), because lack of sequence recognition results in shifts along the DNA axis by one or more base pairs within the same cluster. Instead, the 30 top-ranked placements and the distribution of the 2000 top-ranked placements over the UNG surface were analyzed with computer graphics (Figure 1C and Supplementary Figure S2A).Figure 1.

Bottom Line:
Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264.Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures.These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.

ABSTRACTX-ray crystallography provides excellent structural data on protein-DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein-DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein-DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.